Biomedical subjects

J A Meuwissen

Publications and source records attributed to J A Meuwissen.

At least 19 recordsLinked to original sources

PubMed1992

Testing and characterizing enzymes and membrane-bound carrier proteins acting on amphipathic ligands in the presence of bilayer membrane material and soluble binding protein. Application to the uptake of oleate into isolated cells.

1. A multiphasic modelling approach [Heirwegh, Meuwissen, Vermeir & De Smedt (1988) Biochem. J. 254, 101-108] is applied to systems containing poorly water-soluble amphipathic reactants, membrane material, soluble binding protein and acceptor protein (enzyme or membrane-bound carrier protein). 2. The field of application is constrained by the assumptions (i) that the amount of acceptor-bound substrate is small compared with the total amount and (ii) that all preceding chemical reactions and steps of mass transport are rapid compared with the chemical change monitored. 3. Initial-rate formulae for systems in which an acceptor interacts with unbound or protein-bound ligand are given. The saturation curves are near-hyperbolic or sigmoidal, depending both (i) on the form of ligand (unbound or protein-bound) acted upon by the acceptor and (ii) on whether the assays are performed at constant concentration of soluble binding protein Cp or at constant substrate/binding-site molar ratio RS. 4. Several diagnostic features permit unequivocal distinction between acceptor action on unbound or protein-bound substrate. In the former case, saturation curves, run at the same constant concentration of one of several binding proteins of increasing binding affinity, will show progressively increasing inhibition, the shape changing from near-hyperbolic at Km' less than K1' to sigmoidal at Km' greater than K1'.Km' is the effective Michaelis constant of the acceptor and K1' the effective dissociation constant of the binding sites of the soluble protein (for the sites with the higher binding affinity, if several classes of binding site are present on the protein). Alternatively, the maximum velocity obtained at constant RS less than or equal to 1 should increase hyperbolically with RS/(1-RS) for a binding protein with a single class of binding site. The formula that applies when the binding protein contains two classes of independent binding site is also available. When the acceptor acts on protein-bound ligand, the maximum velocity obtained at constant binding-protein concentration, Cp, increases hyperbolically with Cp. 5. Application of these and additional criteria to initial-rate data on the uptake of oleate into isolated cells supports a mechanism of carrier-mediated uptake of the unbound ligand and allows one to clarify some observations that hitherto had been poorly explained. 6. The influence of soluble binding protein on the reaction and substrate specificities of ligand/acceptor interaction is also discussed. 7. In its present state, data treatment for 'double binding-protein systems' generally requires separate determination of the binding parameters of the soluble binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Carrier Proteins↗

PubMed1989

Modelling of chemical reactions catalysed by membrane-bound enzymes. Determination and significance of the kinetic constants.

A model of multiphasic systems, based on the assumption of zero-order partition of substrates and products into the membranes, is applied to reversible mono-substrate and bi-substrate reactions catalysed by membrane-bound enzymes. Apart from replacement of single-phase kinetic constants by apparent kinetic constants, the derived kinetic expressions are formally identical with those for corresponding single-phase systems. The model confers to the apparent kinetic constants an experimentally verifiable meaning. For full characterization of membrane-kinetic systems, experiments at various concentrations of enzyme-embedding phospholipid are required. Extrapolation to zero phospholipid concentration of each Km app then yields the corresponding true kinetic constant characteristic of the membrane-bound enzyme and also provides a technique for determination of the membrane-partition constants. The procedure implies that the phospholipid content should be assayed for full characterization of membrane-bound enzymes. If, for practical reasons, the assays have to be limited to a single enzyme concentration, correction of the apparent kinetic constants is still possible provided the phospholipid concentration and the partition constants of the reactants are known. The model has permitted prediction of a number of previous observations reflecting the multiphasic nature of the systems. The assumptions, underlying the model, and their implications are examined as well as some commonly used experimental designs for determination of the type of enzymic site.

Binding Sites↗

PubMed1988

Liposomes as carriers of poorly water-soluble substrates: linear modelling of membrane systems with catalytic or binding sites of different facedness. Significance of experimental membrane partition coefficients and of kinetic and equilibrium parameters.

1. A multiphasic modelling approach to systems containing membrane-bound receptors or catalytic sites and a liposomal preparation as a substrate carrier is described. Kinetic expressions are derived for a single-substrate enzymic reaction operating at constant liposome concentration or at a fixed substrate/liposome concentration ratio. 2. The assumption that accumulation of exchangeable components into the phospholipid bilayers can be described by linear bulk-phase partition leads to simple relationships between the initial reaction rate and (a) two kinetic coefficients (V and K'm), (b) the partition coefficients of the solutes for the lipid compartments of the membrane (Pms) and liposomal preparations (P1s) and (c) the total concentrations of substrate, membrane lipid and liposomal lipid. K'm is called the effective Michaelis constant. 3. For correct estimation of the coefficients V, K', Pms and P1s extrapolation to zero lipid concentration is required. 4. The distinction is introduced between hydrophilic and hydrophobic aqueous-faced sites, lipid-faced sites and mixed sites, i.e. sites overlapping an aqueous and a lipid region. For hydrophilic aqueous-faced sites K'm is equal to the true Km and for the other types of site to Km/Ps. For lipid-faced and for mixed sites Ps corresponds to the membrane partition coefficient Pms. For binding of homologous compounds to a hydrophobic aqueous-faced binding pocket Ps is the incremental site partition coefficient Pbss, which takes into account the energetic contribution to the binding process due to the hydrophobic tail of the ligands. 5. K'm accounts for any effects due to the facedness and nature of the enzymic sites. The dependence of the systems on the size of the lipidic partition compartment(s) is expressed exclusively by a distribution function F.6. When enzyme assays are performed with a series of chemically different substrates containing the same catalytically sensitive group, independence of K'm from partition indicates a hydrophilic aqueous-faced binding site. For the low-molecular-mass members of the homologous series a linear increase in -log (K'm) with the logarithm of the partition coefficient will be observed with any of the other site types considered 7. Equilibrium relationships for binding of a ligand to a membrane-bound receptor are also derived. 8. The significance of experimental membrane partition coefficients is discussed.

Binding Sites↗

PubMed1988

Principles of a competitive binding assay for the study of the interactions of poorly water-soluble ligands with their soluble binding partners. Application to bilirubin with the use of Sephadex G-10 as a competitive adsorbent.

1. A generally applicable method is described for obtaining experimental data on the interactions between a poorly water-soluble ligand and soluble binding factors, with the use of chemically inert solid adsorbent. The equilibrium distribution of the ligand between the liquid phase containing the soluble binders and the adsorbent must be measured and knowledge of the binding isotherm of the adsorbent is required. Procedures are given for the calculation of the binding parameters. 2. The method has been applied to quantify the interactions of bilirubin with serum and liver cytosol from the rat, Sephadex G-10 serving as the competing adsorbent. Reversible adsorption keeps the concentration of the free ligand low, thereby preventing formation of colloidal bilirubin. The sensitivity of the procedure accommodates the rather high binding affinities by which bilirubin generally interacts with its specific binding proteins. 3. The binding activities of serum and liver cytosol are of comparable magnitude. Binding of bilirubin by rat serum can be described by two independent binding sites, the affinities of which differ by two orders of magnitude. Only the site with the higher affinity appears to be of physiological importance. The major bilirubin-binding sites of rat liver cytosol seem to contribute equally to the overall binding activity of this preparation, provided that GSH is present.

Adsorption↗

PubMed1987

Selective absorption and scattering of light by solutions of macromolecules and by particulate suspensions.

For particulate suspensions and for solutions that scatter light measurably the total absorbance A generally contains contributions due to specific absorption (Aa) and scattering of light (As). The quantity As is closely related to the turbidity tau. In general, spectrophotometry of such systems requires proper modification of the spectrophotometer used in order to permit accurate determination of the absorbance A and of the derived quantities Aa and As. Apparent deviation from Beer's law in such systems is often due to inappropriate experimental technique. After a discussion of the parameters that determine the intensity of light scattered by solutes, an account is given of the experimental precautions to be taken for determination of the absorbance of light scattering suspensions and solutions and of techniques for correcting absorbance spectra for scattering of light. Measurement of the turbidity is briefly confronted with determination of the scattering ratio i90 degrees/Io and the impact of erroneous turbidity measurements on derived molecular parameters is discussed.

Biopolymers↗

PubMed1979

Ligand binding and detoxication.

In an organism the binding of a toxic chemical to a binding site can act as a detoxication mechanism when toxicity is a property of the unbound ligand. This qualitative statement has been evaluated in quantitative terms. To this end parameters have been defined for which numerical values are required, equations are derived and a procedure is outlined that allows assessment of when and to what extent binding is of value in detoxication. In the process two new quantities are introduced, i.e. the binding capacity and the binding activity, which make for easier handling and comparison of binding data. It is concluded that to be important in detoxication the numerical value of the binding activity must be greater than unity and the total ligand concentration should not exceed the binding capacity. These general conclusions can be further refined depending on the accuracy with which the values of the parameters involved are known. Due to its generality the results of the analysis are useful in all situations where it is desirable to know the magnitude of the free fraction of a bound chemical.

Binding Sites↗

PubMed1977